Tyrissamycin antibiotic

ABSTRACT

A novel tyrosyl diester antibiotic is obtained from fermentation of a recombinant strain of  Streptomyces lividans  designated  Stretomyces lividans  WD 15684 (ATCC-202143). The new antibiotic, designated tyrissamycin, exhibits antibacterial activity, particularly against gram-positive bacteria.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This divisional application claims the benefit U.S.Non-Provisional Application Serial No. 09/378,309 filed Aug. 20, 1999and U.S. Provisional Application Serial No. 60/097,214 filed Aug. 20,1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to a novel tyrosyl diester antibacterialantibiotic designated by the present inventors as tyrissamycin which maybe obtained by cultivation of a strain of Streptomyces lividans. Theantibiotic provided by the present invention has antibacterial activityagainst a variety of gram-positive bacteria.

[0004] 2. Background Art

[0005] The present inventors are not aware of any tyrosyl diestercompound in the literature having a similar structure to tyrissamycin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 shows the ultraviolet absorption spectrum of tyrissamycinin methanol.

[0007]FIG. 2 shows the infrared absorption spectrum of tyrissamycin (KBrpellet).

[0008]FIG. 3 shows the ¹H NMR spectrum of tyrissamycin in CH₃OD.

[0009]FIG. 4 shows the ¹³C NMR spectrum of tyrissamycin in CH₃OD.

SUMMARY OF THE INVENTION

[0010] The present invention provides the novel antibacterial antibioticdesignated by the present inventors as tyrissamycin and a fermentationprocess for production of this antibiotic using a novel recombinantstrain of Streptomyces lividans designated herein as Streptomyceslividans WD 15684 (ATCC-202143). The antibiotic of the present inventionhas been found to be useful for the treatment of a variety of bacterialdiseases caused by gram-positive bacteria.

[0011] Also provided are pharmaceutical compositions of tyrissamycin, amethod for the treatment of bacterial diseases in animals usingtyrissamycin or a pharmaceutical composition thereof, and a fermentationprocess for obtaining the antibiotic.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The tyrissamycin antibiotic of the present invention may beprepared by fermentation of a tyrissamycin-producing strain ofStreptomyces lividans. The preferred producing organism is a novelstrain of Streptomyces lividans TK-64 designated herein as Streptomyceslividans WD 15684. The organism was isolated as a recombinantstreptomycete using the procedures described in published application WO96/34112 and in Chapter 12, pages 71-76, Proceedings from the Geneticsand Molecular Biology of Industrial Microorganisms (GMBIM) Conference,1997, edited by Richard H. Baltz, George D. Hegeman and Paul L. Skatrud.Briefly, DNA was isolated from one or more actinomycetes, none of whichwere Streptomyces lividans. The actinomycete DNA was inserted intoplasmids which were used to transform Streptomyces lividans TK-64. Abiologically pure culture of the resulting recombinant strain WD 15684has been deposited with the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, and added to itspermanent collection of microorganisms as ATCC-202143. The culture isalso maintained as a dormant culture in lyophile tubes and cryogenicvials in the Bristol-Myers Squibb Pharmaceutical Research InstituteCulture Collection, 5 Research Parkway, Wallingford, Conn. 06492. Theresults of taxonomic studies performed on strain WD 15684 confirm thatit is a strain of Streptomyces lividans.

[0013] It is to be understood that for the production of tyrissamycinthe present invention, although described in detail with reference tothe preferred producing strain Streptomyces lividans WD 15684, is notintended to be limited to this microorganism. It is specificallyintended that the invention embrace strain WD 15684 and alltyrissamycin-producing variants and mutants thereof which may beobtained by methods well known to those skilled in the art, eg. bysubjecting the deposited microorganism to x-rays or ultravioletradiation, nitrogen mustard, phage exposure, and the like.

[0014] The tyrissamycin antibiotic may be produced by cultivating atyrissamycin-producing strain of Streptomyces lividans, preferably astrain having the identifying characteristics of Streptomyces lividansATCC-202143, or a mutant or variant thereof, under submerged aerobicconditions in an aqueous nutrient medium containing assimilable sourcesof carbon and nitrogen until a substantial amount of tyrissamycin isproduced by said organism in said culture medium. The tyrissamycin soobtained may be isolated from the fermentation medium in substantiallypure form by conventional procedures such as those described in theexamples which follow. Tyrissamycin exhibits antibacterial activity instandard antibacterial model systems.

[0015] The producing organism is grown in a nutrient medium containingan assimilable source of carbon such as glucose, cellobiose, trehalose,potato starch, glycerol or ribose. The medium should also contain anassimilable source of nitrogen such as fishmeal, peptone, peanut meal,cottonseed meal or cornsteep liquor. Nutrient inorganic salts can alsobe incorporated in the medium so as to provide sodium, potassium,ammonium, calcium, phosphate, nitrate, chloride, bromide, carbonate andlike ions. Trace elements such as copper, manganese, iron, zinc, etc.are added to the medium if desired, or they may be present as impuritiesof other constituents of the medium.

[0016] Production of the tyrissamycin antibiotic may be effected at anytemperature conducive to satisfactory growth of the organism, i.e. 22°C. to 42° C., and is conveniently carried out at a temperature of about32° C. The fermentation may be carried out in shake flasks or inlaboratory or industrial fermentors of various capacities. When tankfermentation is to be used, it is desirable to produce a vegetativeinoculum in a nutrient broth by inoculating a small volume of theculture medium with a slant, a cryopreservative culture or a lyophilizedculture of the producing organism. After obtaining a viable and activeinoculum in this manner, it is transferred aseptically to thefermentation tank charged with production medium for large scaleproduction of tyrissamycin. The medium in which the vegetative inoculumis prepared can be the same as or different from that used in the tankas long as it is such that a good growth of the producing organism canbe obtained. Further agitation may be provided by a mechanical impeller.Antifoam agents such as lard oil or silicone oil may also be added ifneeded. Antibiotic production may be monitored by thin layerchromatography (TLC) assay, TLC bioautographic assay, columnchromatographic assay, high performance liquid chromatography (HPLC)assay or by conventional biological assay.

[0017] When the fermentation is complete, the tyrissamycin antibiotic isextracted from the culture broth with a suitable organic solvent such asethyl acetate or 1-butanol and the antibiotic recovered by the extractand purified by conventional isolation procedures.

[0018] Physico-chemical Properties

[0019] A purified sample of tyrissamycin was isolated as a whiteamorphous powder. Other characterizing properties of the antibiotic areas follows: Solubility: Soluble in chloroform, dimethylsulfoxide (DMSO),and methanol and practically insoluble in water. Ultraviolet Spectrum:UV max MeOH (Absorbtivity): 212 (4.7), 219 (sh. 4.6), 278 (1.6) nm(FIG. 1) Infrared Spectrum: Major Bands (cm⁻¹) 2925, 2854, 1742, 1711,1517, 1465, 1237 cm⁻¹ (FIG. 2) ¹H-NMR: See. FIG.3 (CH₃OD) ¹³C-NMR: SeeFIG.4 (CH₃OD)

[0020] Based on the above properties, the structure of tyrissamycin isbelieved to be

[0021] n₁ and n₂ represent alkylene chains of unknown length

[0022] Biological Properties:

[0023] Tyrissamycin was evaluated in vitro to determine the minimalinhibitory concentrations (MIC) against seven gram-positive bacterialusing the microtiter dilution technique. This method is described by T.B. Conrath, “Handbook of Microtiter Procedures,” Dynatech Corp.,Cambridge, Mass., USA (1972); and T. L. Gavan et al “Microdilution TestProcedures,” in Manual of Clinical Microbiology, E. H. Lennette, Ed.,American Soc. for Microbiol., Washington, D.C., USA (1980). These testsare performed in accordance with procedures established by the NationalCommittee on Clinical Laboratory Standards (NCCLS). The test procedureis as follows: Each well of a sterile 96-well microdilution tray isfilled under aseptic conditions with 0.1 ml of Mueller-Hinton broth. A0.05-ml sample of the test compound solution is added to a well in thefirst row of the tray. A microdilutor apparatus is used tosimultaneously mix the contents of these wells and to transfer aliquotsto each succeeding row of cells to obtain a range of serially dilutedsolutions. The last row of wells is untreated and serves as a control.Each well containing broth and test compound is inoculated with aboutten microliters of inoculum of a given test microorganism. One well inthe last row of wells (which is free of test compound) is not inoculatedand is used as a sterility control. The trays are sealed and incubatedat 37° C. for 16-24 hours. After the incubation period, each plate isevaluated by determining the lowest concentration of test compound thatvisibly inhibits the growth of a given microorganism and recorded as theminimal inhibitory concentration in μg/ml. The antimicrobial activity oftyrissamycin is reported in Table 1. TABLE 1 Antibacterial Activity ofTyrissamycin Minimal Inhibitory Microorganism Concentration, μg/mlStreptococcus pneumoniae 4 Streptococcus pyogenes 4 Enterococcusfaecalis 32 Enterococcus faecium 64 Staphylococcus aureus 32Staphylococcus epidermidis 32 Staphylococcus haemolyticus 32

[0024] As indicated above, the antibiotic of the present invention isuseful as an antimicrobial agent, having utility in inhibiting,including killing, the growth of microrganisms. It is particularlyuseful as an antibacterial agent, especially against gram-positivebacteria such as those of the genera Streptococcus, Enterococcus andStaphylococcus.

[0025] The compound may, for example, be used in a method for treating ahost infected by a bacterium or in preventing infection of said host bysaid bacterium, comprising administering to said host tyrissamycin in anamount effective for said prevention or treatment.

[0026] Hosts include animals, particular mammals such as dogs, cats andother domestic mammals, and, especially, humans. The dosage form andmode of administration, as well as the dosage amount, may be selected byone skilled in the art. The dosage amount will vary with the severity ofthe infection, and with the size and species of the host. Daily dosagesfor an adult human may be determined by methods known to one of ordinaryskill in the art. Administration to a mammalian host may, for example,be oral, topical, rectal or parenteral.

[0027] Pharmaceutical compositions are also provided by the presentinvention which comprise tyrissamycin in an amount effective for theprevention or treatment of infection by a bacterium and apharmaceutically acceptable carrier or diluent. The appropriate solid orliquid vehicle or diluent may be selected, and the compositionsprepared, by methods known to one of ordinary skill in the art. Examplesof such compositions include solid compositions for oral administrationsuch as solutions, suspensions, syrups or elixirs and preparations forparenteral administration such as sterile solutions, suspensions oremulsions. They may also be manufactured in the form of sterile solidcompositions which can be dissolved in sterile water, physiologicalsaline or some other suitable sterile injectable medium immediatelybefore use. The pharmaceutical compositions may contain otherantibacterial agents.

[0028] The compound of the present invention may also be employed as anantimicrobial agent useful in inhibiting the growth of, includingkilling, microorganisms present on a surface or in a medium outside aliving host. The present invention, therefore, provides a method forinhibiting the growth of bacteria present on a surface or in a medium,comprising the step of contacting the surface or medium withtyrissamycin in an amount effective for the inhibition. Thus, theantibiotic of the present invention may be employed, for example, indisinfectants for surface treatment, such as disinfection of surgicalinstruments, or as preservatives for a variety of solid and liquid mediasusceptible to microbial growth. Suitable amounts of the antibiotic maybe determined by methods known to one of ordinary skill to the art.Compositions comprising tyrissamycin in an amount effective forinhibiting the growth of bacteria, and a vehicle or diluent, are alsoprovided by the present invention.

[0029] The following example is provided for illustrative purposes onlyand is not intended to limit the scope of the method.

EXAMPLE 1 Preparation of Tyrissamycin

[0030] General Methods

[0031] Materials

[0032] Hexanes, chloroform, (anhydrous ACS grade), methanol,acetonitrile (anhydrous HPLC grade) and water were not repurified orredistilled. Sephadex LH-20 was purchased from Pharmacia LKB, Uppsala,Sweden.

[0033] Analytical Thin Layer Chromatography (TLC)

[0034] Silica gel precoated thin layer chromatography plates, Kieselgel60 F254 on aluminum sheet, 5×20 cm, 0.2 mm, were purchased from EMSeparations, Gibbstown, N.J. The plates were developed in a tankequilibrated with toluene/ethyl acetate (1:2 v/v). The components of theresulting chromatogram were detected under a UV light, and visualized byphosphomolybidic acid followed by prolonged heating.

[0035] Preparative TLC

[0036] Silica gel precoated Kieselgel 60 F254 plates on glass, 20×20 cm,2 mm, purchased from EM Separations, were used for preparativepurification. The plates were developed in a tank equilibrated withtoluene/ethyl acetate (1:2 v/v). The components of the resultingchromatogram were detected under a UV light. The silica band containingthe components was scraped and pressed to a fine powder, followed byelution with chloroform/methanol (4:1, v/v). The eluant was thenevaporated in vacuo to dryness.

[0037] Analytical HPLC

[0038] The purification of tyrissamycin was monitored by HPLC analysison a Microsorb-MV 5 m C-18 column, 4.6 mm i.d.×25 cm 1. (RaininInstrumnet Company, Inc., Woburn, Mass.). Analyses were done on aHewlett Packard 1090 Liquid Chromatograph, equipped with a modelphotodiode array spectrophotometer set at 254 and 280 nm, and HPLC^(3D)ChemStation operating software. A gradient solvent system and 0.01 Mpotassium phosphate buffer (pH 3.5) was used, according to the method ofD. J. Hook et al (J. Chromatogr. 385, 99, 1987). The eluant was pumpedat a flow rate of 1.2 ml/min.

[0039] Analytical Instrumentation

[0040]¹H-NMR and ¹³C-NMR spectra were obtained on a Bruker AM-500 500MHz instrument operating at 500.13 and 125.76 MHz, respectively, using a5-mm broad-banded probe.

[0041] Extraction and Fermentation of Tyrissamycin

[0042] An aliquot of cryopreserved mycelia from Streptomyces lividansstrain WD15684 is utilized as inoculum for the initial vegetativestagein medium V13 having the following composition: Soluble starch, 2%;glucose, 0.5%; NZ-case, 0.3%; yeast extract, 0.2%; fish meat extract,0.5%; and calcium carbonate, 0.3%. 10 μg/ml of thiostrepton is addedaseptically to each flask after autoclaving to maintain plasmidintegrity. The vegetative culture is incubated at 32° C. and 230 rpm ona rotary shaker for 72-96 hours.

[0043] Following the 72-96 hour incubation, a second set of 500 mlErlenmeyer flasks, each containing medium V13 plus 10 μg/mlthiostrepton, are seeded with 5% of the primary vegetative culture andshaken at 230 rpm for 3 days at 32° C.

[0044] Said flasks are then pooled to ensure uniformity, and 4% to 5% ofthe seed culture is aeseptically transferred to production medium F10A.F10A broth has the following composition: Japanese soluble starch, 2.5%;dextrose, 0.2%; yeast extract, 0.5%; peptone, 0.5%; calcium carbonate,0.3%; and distiller's solubles, 0.5%. 10 μg/ml thiostrepton is addedpost-sterilization.

[0045] The cultures are then fermented on a shaker at 230 rpm for 5days. On the day of harvest, the contents of all flasks are pooled intoa large vessel, pH and sedimentation readings are performed, and thebatch is forwarded to chemistry. The production of tyrissamycin reacheda maximum of about 30 μg/ml at day 5 in the production cycle.

[0046] Isolation of Tyrissamycin

[0047] The extraction and fractionation of the above fermentation brothare monitored by the biological assay described below. Whole broth (20liters) is stirred vigorously with ethyl acetate (6 liters) for twohours. The phases are separated and the organic layer is evaporated invacuo to dryness. The residue (1.35 g) is dissolved in 90% aqueousmethanol (200 ml) and then partitioned against hexane (3×200 ml). Theaqueous methanol layer is diluted with water to a 65% MeOH solution andpartitioned against pre-equilibrated chloroform (3×200 ml). Thechloroform layer is pooled and evaporated to give a crude solid (322mg). To perform column chromatography, the solid is loaded to the top ofa Sephadex LH-20 column and eluted with methanol/chloroform (1:1). Theeluate is collected in fractions of 10 ml and monitored for activity inthe biological assay. The combined active eluate (Fraction 13-17, 130mg) is purified by preparative thin layer chromatography (TLC; SilicaGel 60 F254, Merck, Toluene-Ethyl acetate 1:2) to yield an analyticallypure sample of tyrissamycin (10 mg).

[0048] Biological Assay of Tyrissamycin

[0049] Assays for detection and evaluation of the antibacterial activityof tyrissamycin employ various strains of Staphylococcus aureus. Theprimary biological assay system consists of S. aureus strain SA1.

[0050] Culture Growth and Inoculum Production

[0051] SA1 is grown at 37° C. overnight in Antibiotic Assay Broth (BBL,Cockeysville, Md.). The culture is centrifuged at 4000 rpm for 10minutes and the medium is decanted. Cells are resuspended in 0.9%saline. The culture is adjusted to 25% transmission (600 nm wavelength)using a Coleman Junior Spectrophotometer. This 25% working cellsuspension is stored at 40° C. for use as the assay inoculum.

[0052] Biological Assay Protocol

[0053] All biological assays employ agar diffusion techniques. Assayagar is Seed Agar (BBL) supplemented after autoclaving with 4% ofphosphate-nitrate buffer, pH 7.0, and 0.3% of a 2% solution oftriphenyltetrazolium chloride. For well-agar diffusion assays, assayagar is seeded with 1% of the working cell suspension of SA1. The seededagar is poured into Petri dishes or bioassay trays and allowed toharden. Wells (7 mm diameter) are cut in the agar, and 40 μl samplealiquots are added to the wells. (For some assays, 3 μl of sample arespotted directly on the surface of the agar). Plates are incubated at37° C. for 18-24 hours, and zones of inhibition (if any) are measured.Inhibition zone sizes correlated with the activity of tyrissamycin invarious samples.

[0054] For bioautographic assays, a thin layer chromatograph (TLC) platecontaining a chromatographed sample is placed in a bioassay tray. Seededassay agar is poured over the TLC plate and allowed to harden. Thebioassay tray is incubated at 37° C. for 18-24 hours and examined forthe presence of inhibition zones at a position corresponding to thechemical presence of tyrissamycin.

[0055] Application of the biological assay to the detection andisolation of tyrissamycin is illustrated below.

[0056] Monitoring of Bioactivity During Initial Purification ofTyrissamycin Using a Biological Assay

[0057] Initial purification of tyrissamycin from raw fermentation brothcontaining thiostrepton was accomplished using column chromatographicmethods as described above. Biological assays were performed onFractions 1-31 resulting from said chromatography.

[0058] These procedures yielded a separation of tyrissamycin from otherbiologically active and inactive residues. Results are as follows:Concentration, Inhibition Sample mg/ml Zone, mm* Fraction 1 1 7.0Fraction 2 1 9.0 Fraction 3 1 10.0 Fraction 4 1 16.0 Fraction 5 1 20.5Fraction 6 1 20.5 Fraction 7 1 19.0 Fraction 8 1 15.0 Fraction 9 1 13.0Fraction 10 1 12.0 Fraction 11 1 11.0 Fraction 12 1 9.0 Fraction 13(tyrissamycin) 1 9.5 Fraction 14 (tyrissamycin) 1 10.5 Fraction 15(tyrissamycin) 1 11.0 Fraction 16 (tyrissamycin) 1 10.0 Fraction 17(tyrissamycin) 1 10.0 Fraction 18 1 9.0 Fraction 19 1 8.0 Fraction 20 18.0 Fraction 21 1 8.0 Fraction 22 1 9.0 Fraction 23 1 9.0 Fraction 24 18.0 Fraction 25 1 8.0 Fraction 26 1 8.0 Fraction 27 1 7.0 Fraction 28 17.0 Fraction 29 1 7.0 Fraction 30 1 7.0 Fraction 31 1 7.0

[0059] Monitoring of Bioactivity During Final Purification ofTyrissamycin Using a Biological Assay:

[0060] Final purification of tyrissamycin from column chromatographicfractions containing tyrissamycin was achieved as described above.Biological assays were performed on Fractions 1-4 resulting from saidchromatography. These procedures yielded purified tyrissamycin Resultsare as follows: Concentration, Inhibition Sample mg/ml Zone, mm*Fraction 1 5 7.0 Fraction 2 5 8.0 Fraction 3 5 8.0 Fraction 4(tyrissamycin) 5 14.0

What is claimed is:
 1. A process for the preparation of tyrissamycinhaving the following characteristics: (a) a white amorphous powder; (b)soluble in chloroform, dimethylsulfoxide and methanol, and essentiallyinsoluble in water: (c) exhibits an ultraviolet absorption spectrum whendissolved in methanol substantially as shown in FIG. 1; (d) exhibits aninfrared absorption spectrum (KBr) substantially as shown in FIG. 2; (e)exhibits a proton magnetic resonance spectrum in CH₃OD substantially asshown in FIG. 3; and (f) exhibits a ¹³C magnetic resonance spectrum inCH₃OD substantially as shown in FIG. 4 which comprises cultivating atyrissamycin-producing strain of Streptomyces lividans under submergedaerobic conditions in an aqueous nutrient medium containing assimilablesources of carbon and nitrogen until a substantial amount oftyrissamycin is produced by said organism in said culture medium.
 2. Theprocess of claim 1 which includes the step of isolating tyrissamycinfrom the medium in substantially purified form.
 3. A biologically pureculture of Streptomyces lividans ATCC-202143, said culture being capableof producing the compound tyrissamycin as defined in claim 1 in arecoverable quantity upon submerged aerobic cultivation in an aqueousnutrient medium containing assimilable sources of carbon and nitrogen.